Long QT syndrome (LQTS) is an inherited arrhythmogenic disorder attributable to mutations
in genes contributing to the major cardiac ionic currents, with an estimated prevalence in
industrialized nations of 1 in 2,500 live births (1–3). The cardinal feature of the disease is
a prolonged QT interval on surface electrocardiograms that signals elevated risk for a
life-threatening cardiac arrhythmia with clinical manifestations including palpitations,
syncope, and an increased predisposition for sudden cardiac death (4). Specifically, mutations in KCNH2, which encodes the
rapid component (IKr) of the delayed rectifier potassium channel
(hERG), cause long QT syndrome type 2 (LQT2), which accounts for about 30% of all LQTS
diagnoses (5).

As is commonly observed in many autosomal dominant cardiac channelopathies, the pattern of
inheritance and clinical phenotypes of LQTS patients are complex and often display
incomplete penetrance and/or variable expressivity; some individuals positive for
disease-causing mutations are asymptomatic, and there are varying degrees of phenotypic
severity (6). Specifically, at one end of the
spectrum, mutation-positive individuals within the same family manifest QT prolongation,
repeated syncopal attacks, and sudden cardiac death, while at the other end,
mutation-positive individuals exhibit a relatively normal QT interval with no symptoms
(7). The causes of this variable clinical
expressivity are not well understood. We investigated this question by testing the
hypothesis that modifier genes contribute to the variable clinical expressivity.

The original seminal description of generation of induced pluripotent stem cells (iPSCs)
from somatic cells by Yamanaka and colleagues (8)
ushered in a new era of in vitro disease modeling in myriad fields, including cardiac
channelopathies. This discovery has led to the generation of patient-specific iPSC-derived
cardiomyocytes (iPSC-CMs), which revolutionized cardiac arrhythmia disease modeling (9, 10). While
heterologous expression systems and animal models paved the way for the fundamental
understanding of LQTS pathogenesis, they could not address the variable expressivity seen in
LQTS. However, iPSC-CM–based disease modeling platforms are poised to generate previously
indeterminable mechanistic insights by providing an ultra-versatile in vitro platform that
most closely mimics the native cardiomyocyte setting of the patients.

Furthermore, the impact of modeling cardiac disease in iPSC lines can be synergistically
enhanced with the recent advent of next-generation sequencing technologies such as whole
exome sequencing. While the protein coding regions of the genome constitute approximately 1%
of the human genome (50 million bp), they can account for up to 85% of mutations reported in
Mendelian disorders such as LQTS (11). Historically,
Mendelian diseases such as LQTS are notoriously challenging to diagnose, risk-stratify, and
treat because of their phenotypic heterogeneity and incomplete penetrance. However, the
advancement of whole exome sequencing has exponentially accelerated the ability to screen
for novel disease-modifying genes with base pair resolution (11–13).

For 20 years, an LQT2 family has been studied extensively by our multidisciplinary group
(14, 15).
Initial genotype testing of living family members following the sudden death of a
31-year-old female with a history of syncope along with the subsequent LQT2 diagnosis in a
paternal aunt revealed a C→T nucleotide substitution at position 2,254 in exon 9 of
KCNH2 resulting in the hERG R752W missense mutation. Further genotyping
and ECG testing of 101 family members identified 26 additional hERG R752W mutation–positive
individuals, of whom only 6 have a severely affected phenotype (Supplemental Tables 1 and 2;
supplemental material available online with this article; https://doi.org/10.1172/JCI94996DS1). Previous studies showed that diagnosing
congenital LQTS is difficult because of variable expressivity and genetic heterogeneity.
However, it was posited that a combination of clinical and ECG techniques could identify
gene-positive individuals within a single family with congenital LQTS. Yet, a study
performed on this large Cleveland LQT2 family carrying the hERG R752W mutation demonstrated
that even in this homogeneous LQTS population, the phenotype was so variable that detailed
clinical and ECG analyses did not permit an accurate diagnosis of gene carrier status. In
fact, there was substantial overlap (78%) of QTc among subjects with and without the
mutation, suggesting the presence of 1 or more modifier genes (14). We deployed a novel strategy combining the use of patient-derived
iPSC-CMs alongside exome sequencing and genome editing to unravel novel electrophysiological
arrhythmogenic mechanisms and identify new disease-modifying gene variants that suggest a
plausible explanation for the variable expressivity seen in this LQT2 family. Moreover, this
work showcases the innovative and high-yield approach of using 2 nascent technologies in an
additive manner to unravel the underlying mechanism of familial cardiac channelopathy.

Patient-specific iPSC-CMs recapitulate genotype-phenotype discordance. Two pairs of hERG R752W mutation–positive first-degree relatives (a father/son pair
[III-3 and IV-15, respectively] and a sister/sister pair [IV-3 and IV-4]) with discordant
phenotypes along with a healthy mutation-negative control subject (IV-17) were selected
from this large LQT2 family (Figure 1). iPSC-CMs were
generated from these 5 individuals. We recorded action potential duration
(APD90) from the patient-specific iPSC-CMs to determine whether these
cellular models could recapitulate the genotype-phenotype discordance observed in an LQT2
family. Cells derived from the severely-affected-phenotype father (III-3) exhibited
significantly longer APD in comparison with a mutation-negative first-degree-relative
control (IV-17). By contrast, cells derived from his hERG R752W mutation–positive son
(IV-15) with a mildly affected phenotype did not exhibit a prolonged APD90
despite harboring the same pathogenic hERG mutation (Figure
2A and Supplemental Table
3). Similarly, APD90 recorded from the severely affected sister (IV-3)
was significantly longer than that from her mildly affected sister (IV-4) and the
mutation-negative-relative control, IV-17 (Figure 2B
and Supplemental Table 3).
Notably, APD50 was also significantly prolonged for the 2 severely affected
individuals (III-3, IV-3) compared with their respective mildly affected first-degree
relatives (Figure 2, A and B, and Supplemental Table 3).

Clinical details of carrier pairs in Cleveland LQT2 family. Zoomed-in snapshot of the family pedigree that focuses on 5 individuals of the family
we used to generate the carrier pairs along with their relevant patient history (see
Methods for in-depth explanation of patient selection and phenotype binning criteria and
Supplemental Table 2 for
clinical details on all 26 R752W mutation–positive individuals). Individuals are
referenced first by the generation number and then by the family member number, read
from left to right (e.g., IV-3 is generation 4, family member 3). This reference system
is used throughout the paper. The black square is the hERG mutation negative healthy
male control. Blue circles and squares are females and males, respectively, who are
mildly-affected-phenotype hERG R752W mutation–positive relatives. Red circles and
squares are severely-affected-phenotype hERG R752W mutation–positive relatives. Hatched
circles and squares are females and males, respectively, who are hERG R752W-carrying
individuals who are not fully characterized in this study.

LQT2 genotype-phenotype discordance reproduced in patient-specific
iPSC-CMs. (A) Representative action potential traces from the control (IV-7), a
severely-affected-phenotype LQT2 male (III-3), and his son, a hERG R752W
mutant–positive, mildly-affected-phenotype male (IV-15). Summary APD90 (90%
of repolarization) and APD50 (50% of repolarization) data are also shown
(below traces). (B) Representative action potential traces from the control
(IV-17) and the second pair (sister pair), a severely affected LQT2 female (IV-3) and a
hERG R752W mutant–positive, mildly affected female (IV-4). Summary APD90 and
APD50 data are shown (below traces). (C) Representative
IKr traces and respective summary
IKr tail current density from each patient-derived iPSC-CM
depicted in A. (D) Representative
IKr traces and respective summary
IKr tail current density for each patient-derived iPSC-CM
depicted in B. (E) A 1-Hz paced action potential train from
IV-17 and IV-3 iPSC-CMs. Stars denote early afterdepolarizations. Dashed line in APD
traces denotes 0 mV. Between 30 and 203 cells from 9 different iPSC clones (3 each from
IV-17, III-3, IV-15 paired trio) were analyzed in A and C.
Between 77 and 134 cells from 9 different iPSC clones (3 each from IV-17, IV-3, IV-4
paired trio) were analyzed in B and D. Exact numbers of
replicate measures (n) for each are listed in Supplemental Table 3. Results are
shown as mean ± SEM. *Statistical significance (P < 0.05) as
determined by ANOVA in the summary data for A–D.

We recorded IKr and quantified the tail current density. As
expected, iPSC-CMs from mutation-positive severely affected individuals (III-3 and IV-3)
had significantly smaller IKr density compared with those from
mutation-negative IV-17 (Figure 2, C and D).
Interestingly, mutation-positive but mildly affected individuals (IV-15 and IV-4) also had
significantly smaller IKr tail current density compared with
IV-17 despite not having prolonged APD90. Finally, the recording of paced (1
Hz) action potential trains from III-3 and IV-3 elicited early afterdepolarizations that
were never observed in cells from mildly affected (IV-15, IV-4) or mutation-negative
(IV-17) family members (Figure 2E). This
arrhythmogenic substrate is consistent with an LQT2 phenotype caused by a diminished
repolarization capacity due to reduced hERG current density (16, 17). Collectively, these
data demonstrated the capacity of patient-specific iPSC-CMs to recapitulate the clinical
genotype-phenotype discordance in vitro.

Because the IKr density is similar among all hERG mutation
carriers (Figure 2, C and D), and the APDs between
the control and the mildly affected individual are also similar, this suggests the
presence of compensatory repolarizing currents in the mildly affected individuals. To
assess this, we measured action potential variability in the presence of a hERG channel
blocker, E-4031. Application of E-4031 to all 5 patient iPSC-CM cell lines at 10 and 100
nM prolonged APD90 as expected (Figure 3).
Notably, however, E-4031 at 100 nM significantly exacerbated the existing
IKr deficiency in the severely affected iPSC-CM lines (III-3
APD90 at 100 nM [mean ± SEM], 587 ± 79 ms, vs. no treatment, 368 ± 15 ms; and
IV-3 APD90 at 100 nM, 617 ± 50 ms, vs. no treatment, 413 ± 32 ms).
Comparatively, the mildly affected patient iPSC-CMs did not show the same drastic
prolongation at 100 nM (IV-15 at 100 nM, 291 ± 18 ms, vs. no treatment, 208 ± 14 ms; and
IV-4 at 100 nM, 275 ± 20 ms, vs. no treatment, 186 ± 13 ms), even though the
IKr density was similar between the severely and mildly
affected patient iPSC-CMs (Figure 2, C and D). This
suggests the presence of a compensatory repolarizing current in the mildly affected
individuals.

Effects of IKr blockade on severely and mildly affected
patient iPSC-CMs. (A and B) The effects of E-4031 (hERG channel blocker) at 2
different concentrations on the IV-17, III-3, and IV-15 trio as well as the IV-17, IV-3,
and IV-4 trio. Between 8 and 32 cells were analyzed per E-4031 treatment condition for
APD in A and B. NT, no treatment. *Statistical significance
(P < 0.05) as determined by ANOVA in A and
B.

Patient-specific iPSC-CMs reveal divergent calcium channel activity. Initial electrophysiological analysis of action potential characteristics also revealed a
significant prolongation in the APD50 in iPSC-CMs derived from severely
affected LQT2 patients compared with their respective mildly affected first-degree
relatives and the mutation-negative-relative control IV-17 (Figure 2, A and B, and Supplemental Table 3). A prolongation in APD50 is usually a
reflection of enhanced calcium current. While there were no known clinical indications of
calcium dysfunction in the LQT2 patients, we further investigated this observation by
recording L-type Ca2+ current (ICaL) from the
patient-specific iPSC-CMs. We observed in cells from the severely affected LQT2 patient
iPSC-CMs (III-3 and IV-3) a significantly greater ICaL in
comparison with IV-17 and the mildly affected relatives (IV-4 and IV-15, respectively;
Figure 4, A–C).

L-type calcium current density increase revealed by patient-specific
iPSC-CMs. (A) Representative macroscopic whole-cell L-type Ca2+
(ICaL) traces from the IV-17, III-3, and IV-15 paired
trio. (B) ICaL from the IV-17, IV-3, and IV-4
paired trio. (C) Summary data from A and B.
Between 24 and 95 cells from 9 different iPSC clones (3 each from IV-17, III-3, IV-15
paired trio) were analyzed in A. Between 20 and 47 cells from 9 different
iPSC clones (3 each from IV-17, IV-3, IV-4 paired trio) were analyzed in B.
*Statistical significance (P < 0.05) as determined by ANOVA in
C.

Pharmacological blockade supports Ca2+-mediated LQT2 phenotype. We deployed a pharmacological strategy to further elucidate the contribution of enhanced
ICaL activity in the severely affected patient iPSC-CM
lines. The administration of nisoldipine (a Cav1.2 blocker) at the same
concentration across all cell lines revealed a differential response to Cav1.2
blockade (Figure 5 and Supplemental Figure 1). In the
severely affected patients, 0.05 μM nisoldipine shortened APD90 close to that
of the family control from the same differentiation (Supplemental Table 4). A similar trend
was observed in parallel with the effects of 0.05 μM nisoldipine on L-type calcium current
in the severely affected patients where a larger reduction in current was observed,
bringing the ICaL current density back to the control (Figure 5 and Supplemental Table 4). These results suggest that enhanced
ICaL activity contributes to the action potential
prolongation observed in the severely affected patients.

Nisoldipine reveals increased ICaL sensitivity in
severely affected iPSC-CMs. (A–E) The effect of 0.05 μM nisoldipine on both trios. Data
are depicted as macroscopic action potential record, APD summary data, and
ICaL comparison between no drug and nisoldipine. Between 8
and 11 cells were analyzed for the effects of nisoldipine on APD and 5–8 cells for the
effects of nisoldipine on ICaL in
A–E. See Supplemental Table 4 for exact numbers of replicate measures
(n) and statistical analysis (paired Student’s t
test). Dose-response relationship for nisoldipine (Supplemental Figure 1) was
determined before selection of the concentration used in these experiments (a
concentration lower than EC50 was chosen because the objective was to shorten
the APD and ICaL to levels more like those of control
iPSC-CMs). *P < 0.05; as determined by paired 2-tailed Student’s
t test.

Identification of 2 novel disease-modifying gene variants by whole exome sequencing
and functional electrophysiological interrogation. Since the original report of variable expressivity (14, 15) in this LQT2 family (Figure 1 and Supplemental Tables 1 and 2), we hypothesized that the presence of
disease-modifying gene variants might explain the genotype-phenotype discordance.
Specifically, we postulated that mildly-affected-phenotype mutation-positive family
members (IV-15 and IV-4) were protected from the disease phenotype by a compensatory gene.
Conversely, we hypothesized the existence of a gene variant that exacerbated the primary
hERG mutation in severely-affected-phenotype individuals (III-3 and IV-3). This conjecture
was strongly corroborated by the incidental discovery of increased
ICaL identified in severely affected LQT2 patient iPSC-CM
lines (Figure 4). To investigate these possibilities,
we performed exome sequencing on the same closely related severely affected/mildly
affected phenotype pairs from which we generated iPSC lines.

We used a sequential prioritization strategy in analyzing exome data to identify
potential modifier alleles. This strategy involved dividing the sequenced pairs into
severely affected and mildly affected cohorts, filtering for mutually exclusive
nonsynonymous and small insertion/deletion coding variants, then selecting variants in
cardiac-expressed genes. Special attention was paid to variants in cardiac-expressed ion
channel genes. Next, we filtered for genes identified either by genome-wide association
studies (GWAS) seeking modulators of QT interval duration (18–21) or by the computational network
analysis approach used by Berger and colleagues (22) that grouped genes and nodes into a repository known as the LQTS neighborhood
(Supplemental Figure 2).
Implementing this approach, we identified 12 SNPs and 4 insertion/deletions that could
potentially contribute to genotype-phenotype discordance (Supplemental Table 5) but specifically
pursued 2 gene variants: one found in the mildly affected mutation-positive cohort and one
found in the severely affected mutation-positive subjects.

One variant, KCNK17-p.Ser21Gly, found in mildly affected
mutation-positive individuals (IV-15 and IV-4; Supplemental Table 5) was in a gene (KCNK17) encoding a
2-pore-domain potassium channel (K2p17.1). KCNK17-p.Ser21Gly
is a known common variant with a minor allele frequency of 0.41 in the Exome Aggregation
Consortium (ExAC) database. This was the only variant identified in an ion channel.
Single-cell reverse transcription PCR (RT-PCR) and immunohistochemistry demonstrated
KCNK17 expression in patient-derived iPSC-CMs (Figure 6, A and B). Coexpression of KCNK17-Gly21 and
KCNK17-Ser21 (mimicking the heterozygous state observed in family
members) in Chinese hamster ovary cells revealed significantly greater potassium current
compared with expression of the common allele only (Figure
6, C and D). These findings indicate that KCNK17-p.Ser21Gly is a
gain-of-function variant.

Furthermore, to assess the macroscopic contribution of KCNK17 variants
to APD, we transfected either a KCNK17 siRNA or a scrambled control in 1
severely affected LQT2 cell line (III-3) and 1 mildly affected line (IV-4). Supplemental Figure 3 illustrates the
silencing efficiency of our siRNA on KCNK17 transcript. Action potential
recordings from III-3 (common KCNK17 allele) revealed no difference in
APD in siRNA-treated cells compared with scrambled control (Figure 6, E and F, and Supplemental Table 6). However, KCNK17 suppression in IV-4
(heterozygous for KCNK17-p.Ser21Gly) resulted in significant prolongation
of APD compared with controls (Figure 6, E and F, and
Supplemental Table 6). This
suggests that a KCNK17 gain-of-function variant can be LQTS protective by
promoting APD shortening.

With regard to the severely affected mutation-positive patients, our fundamental approach
was to investigate variants that could potentially explain the unexpected difference in
ICaL (Figure 4)
observed in iPSC-CMs from the severely affected family members. Specifically, we looked
for adaptor proteins or known channel-modulating entities of
ICaL. A common variant in REM2 (p.Gly96Ala;
Supplemental Table 5) with a
minor allele frequency of 0.1 (ExAC) was identified only in our severely affected
mutation-positive subjects. REM2 encodes a member of the
Ras superfamily, which are well-known modulators of voltage-gated
calcium channels. The framework for the identification of this variant was originally
suggested by a computational network analysis of novel genes or “nodes” related to known
congenital LQTS genes (22). In this previous
analysis, a member of the Ras superfamily known as REM
was identified as a candidate gene in what was termed the LQTS neighborhood. Implementing
the LQTS neighborhood as a filter in our own exome sequencing approach and mining our
final data set, we identified REM2, a functionally homologous
ICaL modifier to Rem and also a member of
the Ras superfamily. Thus this gene became a biologically plausible
candidate for explaining enhanced ICaL observed in the
severely affected LQT2 patient iPSC-CMs. Functionally vetting this novel gene variant, we
first demonstrated expression of REM2 in patient-derived iPSC-CMs by
single-cell RT-PCR and immunohistochemistry (Figure 7, A and
B). Overexpressing REM2-p.Gly96Ala in iCell cardiomyocytes
resulted in a significantly greater level of ICaL activity in
comparison with cells expressing the common REM2 allele (Figure 7, C and D). This variant could therefore explain
the enhanced ICaL seen in cells derived from severely affected
individuals (Figure 4).

To further assess the impact of this REM2 variant on APD and
ICaL, we reduced the expression of REM2
using a REM2 siRNA as we did above for KCNK17. Supplemental Figure 3 illustrates the
silencing efficiency of our siRNA on REM2 transcript. Notably, silencing
REM2 had the most significant impact on APD and
ICaL for both severely affected iPSC-CMs that carried the
REM2 variant allele (III-3 and IV-3) (Supplemental Figure 4 and Supplemental Table 7).

Genotyping analysis of variant distribution in hERG R752W mutation–positive family
members. From a translational perspective, we also genotyped an additional 18 patients from this
family. Twelve were hERG R752W mutation carriers diagnosed with varying severities of
LQT2, and 6 were healthy nonmutation carriers. Specifically, we assessed whether the
REM2 variant identified through our exome sequencing strategy
segregated with severely-affected-phenotype patients and conversely whether the
KCNK17 variant distributed in a manner consistent with a protective
role (Table 1). The REM2 G96A
variant was found in all (3/3, 100%) severely-affected-phenotype
individuals but only 3 of 13 (23%) mildly-affected-phenotype patients. Notably, 2 patients
with a mildly affected/minimal phenotype (patients 3 and 6 in Table 1) carry not only the REM2 variant but also the
KCNK17 variant. In these instances, we believe the
KCNK17 variant is able to lessen the severity of the phenotype caused
by the hERG mutation and REM2 variant (as supported by these patients’
mildly affected phenotypes). This strongly corroborates our initial wave of exome
sequencing and electrophysiological data (Figure 7)
indicating REM2 as a modifier gene that exacerbates a preexisting
repolarization deficiency in these patients, particularly in the patients with the most
extreme (severely affected) phenotypes. Conversely, KCNK17 was present in
6 of 13 (46%) mildly-affected-phenotype patients. Importantly, none of the
severely-affected-phenotype patients inherited the KCNK17 variant. This
distribution only in the mildly-affected-phenotype bin also strengthens our rationale and
in vitro iPSC-CM electrophysiological data (Figure 6)
indicating that KCNK17 participates as a protective modifier gene by
mitigating the severity of the primary hERG mutation as a repolarization reserve.
Collectively, the exome sequencing, electrophysiological, and genotyping data are
holistically consistent with REM2 acting as a disease-promoting modifier
gene (especially in the most severely affected phenotypes) and KCNK17 as
a protective gene (as evidenced by the significant distribution rate in
mildly-affected-phenotype individuals).

Here we report the discovery of 2 novel gene variants that provide a physiologically
plausible explanation for variable expressivity in a large subset of patients in a
multigenerational LQTS type 2 family. This discovery was made through the synergistic
pairing of 3 emergent technologies: induced pluripotent stem cell–derived cardiomyocytes
(iPSC-CMs), next-generation whole exome sequencing, and CRISPR/Cas9–mediated genome editing.
To our knowledge this is the first time all 3 platforms have been used in tandem and
corroborated by electrophysiological analyses to elucidate a molecular mechanism of variable
expressivity for a monogenic disorder.

We have confirmed early seminal reports (9, 10) of the capacity of patient-specific iPSC-CMs to
faithfully reproduce disease phenotypes in vitro. While all individuals harboring the hERG
R752W mutation display the same level of IKr deficiency, only
the severely-affected-phenotype individuals had a significant prolongation in APD,
consistent with their clinical phenotype. The similar current density in
IKr observed for severely affected and mildly affected hERG
R752W mutation–positive individuals indicated that the modifier gene(s) was not rescuing
hERG R752W channel expression and was not responsible for the discordance observed in
APD90. However, a serendipitous discovery occurred when we observed a
pathophysiological increase in the amount of L-type Ca2+ current in the
symptomatic patients’ iPSC-CMs even though we had no clinical indication that these patients
had a concomitant calcium channel dysfunction. Importantly, our discovery of abnormal L-type
Ca2+ current would not have been possible in a heterologous expression system
or transgenic animal model. Our use of iPSC-CMs not only recapitulated the known LQT2
phenotype, but exclusively enabled the aberrant calcium current revelation although we had
no prior clinical indications of calcium current abnormality, further validating the unique
power of the patient-specific iPSC-CM platform.

Furthermore, classic efforts to link genes to Mendelian disease are gradually being
displaced by next-generation sequencing platforms (23–25), and exome sequencing has proved
itself as an indispensable technology to identify disease-causing coding variants in
congenital arrhythmia syndromes (26, 27). Thus, we used a strategy seeking to identify gene
variants affecting either ion channels, associated channel proteins, or loci associated with
QT interval variability that could explain variable expressivity in an LQT2 family.
Importantly, since our patient iPSC-CMs revealed a potential calcium dysfunction, we also
hypothesized that this strategy might reveal a mechanistic modifier gene driving the
increased L-type Ca2+ current. This approach led us to the identification and
functional vetting of 2 novel disease-modifying genes.

The first modifier gene we interrogated was KCNK17. Two-pore-domain
potassium channel (K2p) genes such as KCNK17 (encoding
K2p17.1, also known as TASK-4 and TALK-2) are known to be robustly expressed in
myriad tissues, including cardiac and central nervous system, where they traditionally have
been thought to regulate background cell excitability as leak channels (28, 29). However,
a wealth of transgenic animal studies support the notion that TASK family K2p
channels also strongly contribute to cardiac repolarization (30–32). Recently, Friedrich et al. (27) used exome sequencing to identify a gain-of-function
mutation in KCNK17 that in tandem with an SCN5A mutation
underpinned the molecular mechanism of a severely affected arrhythmia phenotype.
Furthermore, work by Schmidt et al. (33) showed that
pharmacological inhibition of a K2p (TASK-1) is a novel potential pharmacological
strategy for treatment of chronic atrial fibrillation. Additionally, Decher et al.
identified a mechanism in a patient whereby increased sodium permeability through a
K2p (TREK-1) predisposed the individual to right ventricular outflow tract
ventricular tachycardia (34). Finally, recent work
from our laboratory corroborates the notion that K2p channels are more than
simply background leak channels, acting as active participants in ventricular repolarization
(35). Collectively, this evidence supports the idea
that K2p channels may modulate cardiomyocyte excitability and contribute to
disease phenotypes as well as providing a therapeutic target. Although our results suggest
that KCNK17 is not a universally protective gene (Table 1), we believe that the p.Ser21Gly variant identified in select LQT2
family members may promote enhanced repolarization reserve that conceivably protects mildly
affected/minimal hERG mutation–positive individuals from a severely affected phenotype, as
supported by 2 individuals (patients 3 and 6 in Table
1) who coinherited the REM2 variant whose deleterious effects are
“canceled out” by KCNK17. Furthermore, in addition to the 2 original
patients in whom we identified the KCNK17 variant via exome sequencing, 2
additional patients (patients 5 and 8 in Table 1)
were categorized as having mildly affected phenotype plausibly attributable to the
protective effect of increased KCNK17 current density. Again, this was
strongly supported by the significant prolongation in APD observed when
KCNK17 was silenced in the individuals carrying the variant with larger
2-pore-domain potassium current but not in the individuals carrying the variant with smaller
2-pore-domain potassium current (Figure 6, E and F). So
while not all mildly-affected-phenotype individuals carried the KCNK17 S21G
variant, our evidence strongly supports the role of KCNK17 as a protective
modifier gene in 6 of 13 LQT2 patients (46%).

The second modifier gene identified in our study is REM2, which belongs to
the RGK family
(Rem/REM2/Gem/Kir) of
GTPases. We pursued REM2 after the fortuitous finding that iPSC-CMs derived
from severely-affected-phenotype patients had significantly enhanced L-type Ca2+
(ICaL) current compared with control and
mildly-affected-phenotype mutation-positive relatives (Figure
4). Cav1.2 channels in the heart are under precise physiological
modulation from a host of intracellular proteins (36). One of these regulators is the Ras superfamily of GTPases
(37), which have been shown to inhibit
voltage-gated calcium channels through putative interactions with auxiliary Cav β subunits
(38, 39).
Experimentally, pharmacological blockade of Cav1.2 using nisoldipine further
revealed the role of ICaL in the patient-specific iPSC-CMs
(which was especially pronounced in the severely affected cell lines; Figure 5). Based on these observations and our iPSC-CM
electrophysiological data, we posited that REM2-p.Gly96Ala identified in
the whole exome sequencing data set explains the robust increase in
ICaL. In fact, expression of this
REM2-p.Gly96Ala variant in control iPSC-CMs (iCells) increased
ICaL to a greater degree than WT REM2 (Figure 7). Furthermore, silencing REM2
using siRNA drastically shortened APD and reduced ICaL in
severely affected iPSC-CMs (Supplemental
Figure 4). More conclusively, the use of CRISPR/Cas9–mediated genome editing to
correct the REM2 G96A mutant allele to wild type in the severely affected
IV-3 iPSC line returned the APD and ICaL to near control-like
levels (Figure 7, E–H, and Supplemental Table 8). Finally, the
additional genotyping of 18 family members strongly corroborates this variant as a plausible
explanation of variable expressivity in a subset of family members. Similarly to
KCNK17, while REM2 is not the universal driver of
disease in this family, we show here that it segregates with 100% (3 of 3) of
severely-affected-phenotype LQTS patients in this family (Table 1). Collectively, the experimental and patient observations confirm the
physiological role of REM2, a member of the Ras
superfamily of GTPases, which has previously been described to regulate voltage-gated
calcium channels (39, 40). And while the seminal report of Splawski et al. (41) originally reported and linked CACNA1C mutations to
Timothy syndrome (with long QT syndrome type 8 overlap), here we report a novel role for a
known voltage-gated calcium channel regulatory protein to exacerbate a canonical LQT2
mutation. To our knowledge this is the first description of a voltage-gated calcium
channel–associated protein behaving as a modifier gene for LQT2. But importantly, it is also
the first report implicating REM2 as a regulator of cardiac
electrophysiology, making it a potential new LQT gene.

The hypothesis of a “double hit” that exacerbates or results in a proarrhythmic phenotype
as described in this report is neither unprecedented nor unfounded. While historically LQTS
is considered a “one gene, one disease” etiology, the genetic landscape is quite complex,
and evidence is mounting for more oligogenic or oligo-SNP–based drivers of disease. The
compounding effects of 2 mutations in the same gene (e.g., compound heterozygosity) or 2
separate genes (digenic heterozygosity) are well documented in not only LQTS (42, 43), but other
cardiac pathologies as well (44–46). Consistent with the findings in these reports, we propose here a new
take on the double hit hypothesis: a primary mutation in a gene that dictates the bulk of
ventricular repolarization (hERG) in tandem with an SNP in a gene that encodes a
voltage-gated calcium channel–associated protein (REM2). This combination
results in exacerbation of a primary deficiency in repolarization responsible for the final
disease phenotype. We also show that a second SNP in KCNK17, which encodes
a 2-pore-domain potassium channel, can protect patients by behaving as a compensatory
current in the presence of a primary repolarizing deficiency (including 2 patients who have
both a primary hERG mutation and the REM2 SNP, in which case the
KCNK17 SNP serves as the “tie breaker”).

Study limitations. While various other modifier gene candidates were also identified through our exome
sequencing prioritization strategy, we elected to focus on 2 rapidly assayable and
biologically plausible targets that could directly influence QTc and explain the increase
in ICaL from severely-affected-phenotype mutation-positive
patients. Other candidates were excluded because either their primary functions are
described in a noncardiac setting (e.g., GRIN3A, ref. 47; and CAMKK2, ref. 48) unrelated to calcium channel function or these
variants were deemed nonpathogenic by in silico predictors (PolyPhen, SIFT). Conceptually,
our exome sequencing filtering approach was designed to identify modifier genes in an LQT2
family and not de novo mutations. KCNK17 was pursued because it is the
only ion channel that turned up in either cohort, and REM2 is a known
modulator of voltage-gated calcium channels that could explain the increase in
ICaL observed in Figure
4. However, we do not exclude the likelihood of some permutation of these other
variants identified in Supplemental Table
5 to also contribute to either the protective effect in the mildly affected
individuals or the additive effect in the severely affected individuals. In fact, this is
probably the cause for individual 7 in Table 1,
who, even though he carries the hERG mutation and the REM2 variant,
displays a mildly affected phenotype. However, pursuing the identification of additional
modifiers in an efficient and clinically translational approach will be the focus of
further studies.

Additionally, while filtering using GWAS runs the risk of missing rare variants, we used
this filter to ensure that we cast a wide enough net so as to not exclude previously
identified common modulators of QTc. Another consideration with these diseases is that
while the mutations set baseline risk, the exact timing of the lethal events depends on
triggering factors yet to be identified, suggesting that the variable expressivity could
be ascribed to environmental or other acquired influences. However, it has been clinically
observed with this specific family that in carriers with multiple ECG recordings
documented over 20 years, variation in QTc with time was minimal (14). Hence, the variable expressivity of QTc in these family members
carrying the same mutation with stable QTc values over time suggests an important
influence of modifier genes with environmental factors playing a minor role. Here, we have
not identified the universal mechanism of disease expressivity in this family, but rather
isolated and vetted 2 disease-modifying genes that provide a plausible explanation for
increased or reduced risk of severely affected LQT2 phenotype.

Conclusions. In summary, we demonstrate the synergistic usage of iPSC technology, next-generation
exome sequencing, and CRISPR/Cas9 genome editing to identify plausible genetic
explanations for variable phenotypic expressivity in a large LQTS type 2 family. We
postulate that REM2-driven increased ICa,L in
combination with a primary KCNH2 (hERG, IKr)
haploinsufficiency is the permutation that produces the full-blown disease phenotype and
that KCNK17 can promote a compensatory repolarization reserve.
Importantly, we have uncovered 2 new genes that could be linked to arrhythmias. Finally,
we advance that this strategy can be deployed to screen for novel genetic modifiers in
at-risk populations extending beyond the cardiac channelopathy landscape. All 3
technologies accelerate the efficiency and resolution of informing on candidates
underlying genotype-phenotype discordance. We show that the combinatorial usage of these
platforms can pinpoint novel and potentially actionable targets, thus laying the framework
for a tractable approach to precision medicine.

Patient IRB approval and informed consent. Study subjects were ascertained following written informed consent procedures approved by
the Institutional Review Board of Case Western Reserve University and in accordance with
the MetroHealth Medical Center Human Investigation HIPAA Authorization Policy.
Comprehensive clinical analyses in the enrolled family members (white, n
= 101) include history, physical examination, and ECG recording.

Selection of patients in the study and clinical diagnosis parameters. Patients described in this study were initially identified and diagnosed by a clinical
cardiac electrophysiologist who has been tracking and treating this family for 20 years
and previously reported on the variable expressivity of LQTS in this family (14). Furthermore, molecular work has previously
elucidated the disease-causing mechanism for the hERG R752W mutation (15). Phenotype binning (severely affected and mildly
affected) was based on previously determined clinical diagnostic criteria (ECG morphology
abnormalities and arrhythmic syncope).

Two pairs of hERG R752W mutation–positive severely-affected-phenotype versus
mildly-affected-phenotype first-degree relatives (a father/son pair [III-3 and IV-15,
respectively] and a sister/sister pair [IV-3 and IV-4]) along with a healthy
mutation-negative control subject (IV-17) were selected from this large LQT2 family (Figure 1). We proceeded with these individuals to drive
the crux of the study based on their close genetic relationship, but divergent clinical
phenotypes.

To control for intraclone variability, multiple clonal lines were generated per patient
described in Figure 1. These lines were stored and
continuously propagated and used for cardiomyocyte differentiation and
electrophysiological characterization.

Preparation of iPSC lines for cardiomyocyte differentiation. iPSC vials were thawed from liquid nitrogen storage and cultured on 6-well dishes that
were coated with Matrigel prepared according to company protocol (Corning Matrigel
hESC-qualified Matrix). Cells were pelleted by centrifugation and resuspended using 6–12
ml of mTeSR medium (STEMCELL Technologies) plus 5 mM ROCK inhibitor (StemRD) and plated on
6-well Matrigel-coated dishes. mTeSR medium was changed daily until iPSCs were ready for
passing (5–8 days after plating). iPSC colonies that achieved confluence were incubated
with Accutase (Stem Cell Technologies) to generate smaller cell aggregates. Cell scrapers
were used to gently detach the colonies, which were centrifuged and dissociated by
pipetting, then plated onto Matrigel-coated dishes in dilutions of 1:3 to 1:6.

In vitro cardiomyocyte differentiation from iPSCs. Human iPSC lines were differentiated to cardiomyocytes using a previously described
Wnt-based high-yield protocol (50, 51) and lactate purification step (52). Briefly, iPSC colonies were washed with 1 ml of
PBS, and 1 ml of TrypLE Express (Gibco) was added to each well. Colonies were incubated in
trypsin and dissociated to single cell by slow pipetting. Cell suspensions were pelleted
by centrifugation (3×), then counted manually (Neubauer chamber). Singularized cells were
seeded into Matrigel-coated 12-well dishes in concentrations of 500,000, 750,000, and 1 ×
106 cells per well. Cells were cultured for 4 days, and monolayers were
incubated in 2 ml solution of RPMI medium (Invitrogen) enriched with B27-Insulin
supplement (Invitrogen) plus 12 μM CHIR99021 (Selleck) in DMSO for 24 hours (considered
day 0). Twenty-four hours after addition of CHIR99021 molecule, medium was replaced with 2
ml of RPMI + B27-Insulin supplement. On day 3, 1 ml of the old medium from each monolayer
was mixed with 1 ml of fresh RPMI + B27-Insulin supplement plus 5 μM IWP4 (Stemgent). On
day 5, monolayers were expanded and washed with PBS (1 ml/well) to rinse IWP4 and cellular
debris. Accutase was added to the cells, monolayers were titrated to singularize cells,
and cell suspension was pelleted by centrifugation. Each monolayer was split 1:3 into
fibronectin-coated dishes. Usually, cells started spontaneously beating at day 8. Cells
were fed every 2 days with RPMI + B27 until day 20, when lactate purification was used as
a metabolic stressor. Dead cells were flushed, and around day 28, beating cells were ready
for harvest/dissociation and plating for electrophysiological analysis. Immunocytostaining
of patient-derived iPSC-CMs for canonical sarcomeric proteins confirmed cardiomyocyte
lineage (Supplemental Figure
5).

Mitigation of iPSC-CM cellular phenotype variability. iPSC-CMs can exhibit large variability in APD between differentiations (e.g.,
interbatch). To mitigate this known phenomenon, our analysis is performed in a manner in
which we never compare differentiations from different time points or from different
batches. In other words, a control cell line (IV-17) is always differentiated
concomitantly with a severe and mild iPSC-CM cell line, and all data collected are
analyzed as a trio. Cross-comparison across groups and differentiations is never performed
because each differentiation has intrinsic and systemic differences that must be uniformly
maintained within the trio. Thus, each time we have compared data within a trio, we have
always observed the same results: significantly prolonged APD for the severely affected
individual compared with the control or mildly affected pair (although from
differentiation to differentiation the absolute APD values might have varied). Using this
methodology, we have observed a conservation of the observed trends over multiple
differentiations over a 5-year period.

CRISPR/Cas9 genome editing. Genome editing of cell lines was contracted through Applied Stem Cell. The
REM2 A96 variant in IV-3 iPSC cells was converted to G96 using the
CRISPR/Cas9 system. Briefly, guide RNAs (gRNAs) were designed and tested as transfection
candidates based on the following criteria: (a) an assessment of gRNA activity as measured
by deep sequencing; and (b) proximity to the corresponding point mutation insertion site.
Based on off-target analysis, 2 gRNAs were chosen: g7 [EA102.REM2.g7 TGAACTTGACTGGCCACCTC
(AGG)-PAM] and g12 [EA102.REM2.g12 CGTCTGACTCCTTGGGCTCA (GGG)-PAM), where next-generation
sequencing data show 45% and 72% of gRNA activity, respectively. Each of the
single-stranded oligodeoxynucleotide donors (ssODNs) was designed to be used as a repair
template at each of the gRNA cut sites during the homology-directed repair process (ssODN
sequence:
CAGACGAAGAGGCAGTATGCCTGTCCCCTACAAGCACCAGCTCCGGCGGGCCCAGGCTGTAGATGAACTTGACT GGCCACCTCAGGCCTCATCCTCTGGCTCGTCTGACTCCTTGGGATCCGGAGAGGCAGCCCCTGCTCAAAAGGAT GGCATCTTCAAGGTCATGCTAGTGGGGGAGAGCGGCGTGGGCAAGAGCAC).
After electroporation of both gRNAs into the iPSCs, and since the PGK/Puro construct
provided transient puromycin resistance, the transfected iPSCs were subjected to puromycin
selection for 2 days after transfection; then single-cell colonies were grown for another
few weeks. Single-cell colonies were picked and transferred into a 24-well plate for
further growth. After colonies reached 90% confluence, cells were divided into 2 parts, 1
part was sent for genotyping, and remaining cells were seeded into the newly prepared
24-well plate with Matrigel. Screening indicated that 2 clones contained the predicted
digestion pattern of Gly96 homozygous mutation. Sequence analysis of these clones
confirmed the change of GlyAla96Gly (GCC→GGC). Differentiation of corrected iPSCs to
cardiomyocytes was performed as described above under In vitro cardiomyocyte
differentiation from iPSCs.

Electrophysiological assessments. Electrophysiological recordings in the voltage-clamp and current-clamp modes of the
whole-cell patch clamp (53) were obtained using an
Axopatch 200A amplifier and acquired with a Digidata 1440A digitizer. To generate
voltage-clamp command pulses, pCLAMP version 10 was used (Molecular Devices). To minimize
voltage-clamp errors, series resistance compensation of Axopatch 200A was performed to
values greater than 85%. Patch electrodes were prepared from 8161 Corning borosilicate
capillary glass (Dow Corning) and lightly polished to 1.5–2.5 MΩ for iCells, iPSC-CMs, and
Chinese hamster ovary cells. The amphotericin-perforated patch technique was used to
obtain whole-cell recordings of action potentials under current clamp conditions.
Whole-cell voltage-clamp recordings of IKr,
IK2p17.1, and ICaL were obtained
using standard patch clamp technique. Action potentials and
IKr were recorded at 37°C, whereas all other currents were
recorded at 21°C. All ionic currents were normalized to cell capacitance. The cells were
bathed in a chamber continuously perfused with Tyrode’s solution composed of (in mmol/l
adjusted to pH 7.35 with NaOH) 137 NaCl, 5.4 KCl, 2 CaCl2, 1.0
MgSO4, 10 glucose, 10 HEPES. The intracellular pipette solution was composed of
(in mmol/l adjusted to pH 7.3 with KOH) 120 aspartic acid, 20 KCl, 10 NaCl, 2
MgCl2, 5 HEPES, and 240 μg/ml of amphotericin B (Sigma-Aldrich). A gigaseal
was rapidly formed. Typically, 10 minutes later, amphotericin B pores lowered the
resistance sufficiently to current-clamp the cells. Myocytes were paced in the
current-clamp mode using a 1.5- to 2-diastolic-threshold 5-ms current pulse at 1 Hz.
Action potential trains were also recorded at 1 Hz and further analyzed using Clampfit
software. The extracellular bath solution and intracellular pipette solution used for
IKr were the same Tyrode’s as used for action potentials.
The extracellular bath solution for ICaL contained (in mmol/l
at pH 7.4 adjusted with HCl): 50 NaCl, 5.4 CsCl, 1.8 MgCl, 1.8 CaCl2, 10 HEPES,
10 glucose, 80 N-methyl-D-glucamine. The intracellular pipette solution contained (in
mmol/l at pH 7.2 adjusted with CsOH): 130 cesium methanesulfonate, 20 tetraethylammonium
chloride, 1 MgCl2, 10 EGTA, 10 HEPES, 4 Mg-ATP. This solution was used to
record ICaL from iCell cardiomyocytes as well as iPSC-CMs. The
extracellular bath solution for IK2p17.1 contained (in mmol/l
at pH 7.4 adjusted with NaOH): 140 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2,
10 HEPES, 10 glucose. The intracellular pipette solution used for
IK2p17.1 (KCNK17, 2-pore-domain
K+ current) contained (in mmol/l at pH 7.2 adjusted with KOH): 100
K-aspartate, 20 KCl, 2 MgCl2, 1 CaCl2, 10 EGTA, 2 ATP, 10 HEPES.
IKr was elicited by 3-second depolarizing steps from a
holding potential of –40 mV to potentials ranging from –20 to +20 in 20-mV increments.
This was followed by a 2-second repolarization phase to –40 mV to elicit tail current.
ICaL was elicited by 300-ms depolarizing steps from a
holding potential of –40 mV to potentials ranging from –30 mV to +60 mV in 10-mV
increments. IK2p was elicited by 500-ms depolarizing steps
from a holding potential of –80 mV to potentials ranging from –120 mV to +80 mV.

Enrichment, library preparation, and exome sequencing. Whole exome sequencing was performed on genomic DNA from the 2 first-degree relative
pairs (pedigree generation and family member numbers: III-3, IV-15; and IV-3, IV-4) of
mutation-positive subjects by the Vanderbilt Technologies for Advanced Genomics core
facility. Exome enrichment was performed using the Agilent Sure Select Human All Exon 50
Mb capture reagent according to the supplier’s instructions. Paired-end (2 × 100 bp)
sequencing was performed on an Illumina HiSeq2000 sequencer.

Exome sequence analysis. After removal of low-quality reads, alignments to a reference human genome (UCSC Genome
Browser assembly hg19) were performed with the Burrows-Wheeler Aligner (BWA) (11), and then sequences were processed with the Genome
Analysis Toolkit (Broad Institute) to remove duplicate reads and to call variants. Default
settings were used in the BWA alignments, including a maximum of 2 mismatches in the seed
portion of reads (first 32 bp) and no more than 3 mismatches for the entire read. The
threshold for detecting variants was set at a genotype quality score of 40.

Generation of KCNK17 and REM2 mutations. The REM2 plasmid was provided by Henry Colecraft (Columbia University,
New York, New York, USA), and the KCNK17 plasmid was provided by Dierk
Thomas (Heidelberg University, Heidelberg, Germany). The REM2 G96A and
KCNK17 S21G variants were created using Stratagene QuikChange XL
Site-Directed Mutagenesis Kit in the REM2 background (PubMed accession
no. NM 173527) expressed in the pCDNA4 vector (BD Biosciences Clontech) and the
KCNK17 background (PubMed accession no. NM 031460) expressed in the
pCDNA3.

Expression of KCNK17 in Chinese hamster ovary cells and REM2 in iCell
cardiomyocytes.KCNK17 and REM2 were expressed using transient
transfections in Chinese hamster ovary (CHO) cells or iCell cardiomyocytes (Cellular
Dynamics International), respectively. The total DNA transfected was equal to 2.0 μg for
both conditions (1.8 μg of KCNK17 or REM2 DNA + 0.2 μg
of yellow fluorescent protein). In the case of the KCNK17 heterozygote
condition in CHO cells, the total DNA transfected was still 2.0 μg but divided evenly at a
1:1 ratio (0.9 μg per condition + 0.2 μg of yellow fluorescent protein). Transfection of
both CHO cells and iCells was accomplished using Lipofectamine 2000 (Life Technologies)
according to the manufacturer’s protocol, using 2.0 μg DNA and 35-mm dishes (CHO cells) or
scaled for a 24-well plate (iCells).

KCNK17 and REM2 siRNA knockdown experiments. iPSC-CMs from patients were transfected with either a TALK-2 (KCNK17)
siRNA (Santa Cruz Biotechnology, sc-61641), a REM2 siRNA (Santa Cruz
Biotechnology, sc-92154), or Ambion siRNA scrambled negative control (Ambion/Life
Technologies) using Lipofectamine RNAiMax (Life Technologies) at 20 nM of final
concentration in a 24-well plate. Cells were also cotransfected with 10 nM of Ambion
FAM-labeled siRNA negative control (Ambion/Life Technologies) to identify transfected
cells. Action potentials from transfected cells were recorded 48 hours after
transfection.

REM2 and KCNK17 genotyping. Genomic DNA was extracted from whole blood and REM2 and
KCNK17 were sequenced in 18 family members from the LQT2 family. Exon 2
of REM2 and exon 1 of KCNK17 were sequenced in these
individuals using specific primers (REM2 forward primer
ACAGCCTGGATTGAGCCTTC, reverse primer CAGGTCACGAGCACTTCCTT; KCNK17 forward
primer CCATTCCCCAACACTCCTCC, reverse primer CGAGGGTCTTTTCCTCCGAG). PCR reactions were
performed using 50 ng of genomic DNA with primers corresponding to exon 2 of
REM2 and exon 1 of KCNK17. PCR products were sent out
for sequencing (Eurofins MWG Operon).

Single-cell RT-PCR. Single-cell reverse transcription PCR (RT-PCR) was performed by patch-clamping of the
cell to obtain the electrophysiological verification of myocyte type by action potential
recording followed by harvesting of the cell via extended suction using the same pipette.
The contents of the pipette were deposited into a tube containing a scaled reverse
transcription mixture (High Capacity Reverse Transcription kit, Applied Biosystems).
Following reverse transcription, PCR was carried out using 2 μl of the RT reaction.

Statistics. All statistical analysis was performed using the standard statistics suite available in
Origin 8.5.1 (OriginLab). Normality and homogeneity of all data sets were determined using
the Shapiro-Wilk and Levene tests, respectively, and by visual inspection of distribution
in our data sets. Multiple comparisons between severely-affected-phenotype LQT2
mutation–positive individuals and mildly-affected-phenotype mutation-positive individuals
or non-hERG-mutation-carrying controls were performed using ANOVA. E-4031 and nisoldipine
experiments were analyzed using the paired Student’s 2-tailed t test.
Two-sided P values less than 0.05 were considered statistically
significant. All results are reported as mean ± SEM unless otherwise noted.

ESK performed clinical assessment and referred the family. SC, EF, ALG, ESK, and ID
contributed to study conceptualization and experimental design. SC and XW performed in vitro
data collection. ARN and PJT performed iPSC generation and cardiomyocyte differentiation.
ALG performed the exome sequencing. SC, ALG, ESK, and ID wrote and edited the
manuscript.

We thank Henry Colecraft for providing the REM2 plasmid and Dierk Thomas
for providing the KCNK17 plasmid. This study was supported by NIH/NHLBI
grant 1R01HL124245 (to ID), American Heart Association Established Investigator Award
12EIA9300060 (to ID), an American Heart Association Pre-Doctoral Fellowship from the Great
Rivers Affiliate 15PRE25700037 (to SC), and T32 Training Grant HL105338-01 (to SC). This
publication was made possible by the Clinical and Translational Science Collaborative of
Cleveland, grant UL1TR000439 from the National Center for Advancing Translational Sciences
component of the NIH, and the NIH Roadmap for Medical Research. Its contents are solely the
responsibility of the authors and do not necessarily represent the official views of the
NIH.